STREPTOCOCCAL L FORMS

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1 STREPTOCOCCAL L FORMS I. EFFECT OF OSMOTIC CHANGE ON VIABILITY' CHARLES PANOS AND S. S. BARKULIS Department of Biological Chemistry, University of Illinois College of Medicine, Chicago, Illinois Received for publication February 6, 1959 An L form was first isolated by Klieneberger in 1935 from cultures of Streptobacillus moniliformis and classified as a symbiont of the streptobacillus (Klieneberger, 1935). It was subsequently shown by Dienes (1938, 1939) and Dawson and Hobby (1939), however, that the L form was a growth form of the bacillus and not a symbiont. Since the initial isolation, L forms have been obtained with regularity from a number of bacterial genera. The techniques for obtaining, handling, and cultivating this form have been amply detailed in excellent reviews (Dienes and Weinberger, 1951; Klieneberger-Nobel, 1954; and Kandler and Kandler, 1954) and in a paper by Sharp (1954). A characteristic of L forms, recognized since their earliest study, is fragility. It was this property which led to the conclusion that this form lacked a rigid cell wall. Proof for the absence of a rigid cell wall was provided by a number of methods. Electron microscopic examination revealed the presence of a "veil-like" membrane in place of the easily discernible thick and rigid cell wall structure (Smith et al., 1948). Penicillin, whose mode of action is purported to be inhibition of cell wall formation, was without effect on the growth of this form. More conclusively, Sharp et al. (1957), in their examination of L forms from various group A streptococci for cell wall constituents, were unable to demonstrate the presence of rhamnose and hexosamine which make up the group specific polysaccharide of the cell wall. They suggested that the lack of rigidity in the L form was due to the absence of the cell wall polysaccharide. L forms from #-hemolytic streptococci were successfully isolated recently with the aid of penicillin and high electrolyte concentrations (Sharp, 1954). This paper deals with the effect of osmotic change on the viability of an L form from a group A, f-hemolytic streptococcus. Some of these results have been reported in preliminary form (Panos and Barkulis, 1958). MATERIALS AND METHODS Growth of the organism. The L form employed in these investigations was obtained from Dr. Louis Dienes or was reisolated from the parent organism, a nontypable group A, 3-hemolytic streptococcus designated AED, as previously described (Sharp, 1954). The liquid medium utilized for growth was composed of 2.8 per cent brucella broth (Albimi), 3 per cent NaCl, 10 per cent horse serum inactivated at 56 C for 30 min, and penicillin (1000 units per ml). For the solid medium, 4 per cent trypticase soy agar (BBL) served in place of the brucella broth. Horse serum and penicillin were added separately to the medium after it had been adjusted to ph 7.0 to 7.2, autoclaved for 10 min, and cooled. In all experiments, L form cultures and suspensions were examined by the stained agar preparation technique (Dienes, 1945; Dienes and Weinberger, 1951), phase microscopy, and with a double lens magnifier (3.5 X). Optical density readings as an index of growth were obtained with a Beckman model DU spectrophotometer set at 450 my and only 16 to 20-hr cultures with optical density readings between and were used. All test solutions in which the L form was suspended were prepared on a weight/volume basis, 1000 units per ml of penicillin added, the ph adjusted to 7.2, and sterilization effected by Berkfeld filtration. MgSO4-7H20 served as the source of Mg++ and 0.3 M phosphate buffer was - prepared with K2HPO4 3H20 and adjusted to ph 7.2. Reagent grade chemicals were employed in all solutions. 1 This investigation was supported by a grant RESULTS (no. E1514) from the Institute of Arthritis and Infectious Diseases of the U. S. Public Health Growth characteristics of the L form in liquid Service. media. Figure 1 illustrates the growth curve of 247

2 248 PANOS AND BARKULIS [VOL. 78 c 0._ I I H o u r s Figure 1. Growth curve of AED L form. the streptococcal the streptococcal AED L form. This strain had been subcultured at least 90 times on agar and in liquid media. Although visible growth occurred after 20 hr on agar, broth cultures usually attained their maximal cellular mass after only 8 hr incubation. Dienes has previously described the difficulty of initiating growth of the L form in liquid media. We have observed that once growth was established in a 10 per cent horse serum broth medium, subculturing became less difficult. The growth rate increased until there was no significant difference between the rate of growth of the L form and the parent streptococcus. Horse serum as well as brucella broth was necessary for growth. On omission of either constituent, no growth was observed even after prolonged incubation. Changes in morphological characteristics also developed upon continued subculture in a liquid environment. Upon isolation and initial growth in broth the macroscopic appearance of this L form resembled individual small and granular "ball-like" structures which quickly settled to the bottom of a flask after agitation. Microscopically, the intact L structure varied in size and was composed of aggregates of intact large bodies of differing size and density. In some of these dense large bodies, the reproductive granular elements described by Dienes were evident. Very few of the large bodies were found free in the suspension. After prolonged subculture in liquid media, i. e., at least 60 subtransfers, the macroscopic appearance of the L form became progressively typical of most bacterial cultures. Growth no longer appeared as small ball-like aggregates but was homogeneous and showed sedimentation only after prolonged incubation. Microscopically, the intact L structure was not as prevalent or as large as in the original broth culture. Instead, the structure had almost completely dispersed, with the various components floating freely in pairs, or small aggregates throughout the medium. Although reaggregation occurred upon storage at 4 C, dispersion was easily accomplished with slight agitation. A photomicrograph of stained preparations of the L form from a broth culture appears in figure 2. The characteristic L form morphology displaying the dense large bodies is clearly discernible. Growth after suspension in various solutions of differing osmolarity. A simple method for assessing the fragility of the L form was to determine its viability after exposure to solutions of varying osmolarity and composition. Three-ml samples of the culture were centrifuged at 2200 rpm in an anglehead centrifuge (Servall, SS-1) for 20 min in 16 by 120 mm test tubes and the medium aseptically decanted. Following resuspension and recentrifugation, twice with 3-ml samples of the respective test solutions, the L form was resuspended in a third sample and allowed to remain at room temperature (25 to 27 C) for 5 hr. After examination by dark phase microscopy and following centrifugation, the test solution was decanted and the cellular mass transferred into a 50-ml Erlenmeyer flask containing 10 ml of the liquid medium. The optical density was recorded after 20 to 22 hr incubation at 35 C. Incubation was continued for 72 hr for those cultures displaying no growth after 20 to 22 hr. Figure 3 illustrates the growth of the L form following suspension in various solutions. There was no significant difference in the amount of growth obtained after suspension in solutions containing equal molar concentrations of sucrose or phosphate (IX-XIII, VI). Growth following suspension in 0.3 M phosphate buffer (VI and XIII), however, proved to be erratic. Consistent

3 0 ;: X;. S. e.... W. : : :.:...: :: ] VIABILITY OF STREPTOCOCCAL L FORMS 249 AW;Xt' w : ::.:: ::..... *: i Figure 2. Photomicrograph of the L form from a broth culture by the stained agar block technique of Dienes (magnification, X980). results were difficult to reproduce with the incubation, solutions without sucrose or phosgrowth response varying from 10 to 100 per cent phate buffer (I-V) did not respond even after of the refrigerated control after 20 to 22 hr these prolonged incubation periods. In the incubation. Although maximal growth with these various sucrose concentrations tested (0.1, 0.15, solutions usually developed after 48 to 72 hr 0.2, 0.25, 0.3 (XII), 0.5, and 0.88 M) growth...

4 250 PANOS AND BARKULIS [VOL ~ m x XE Xs x Figure S. Growth of the L form following suspension in various solutions. I. Distilled water, penicillin; II. Distilled water, without penicillin; III. Buffered saline (0.007 M Na2HPO4-NaH2PO4, ph 7.3); IV. Normal saline (0.15 M); V. Mg++ (0.008 M); VI. Phosphate buffer (0.3 M, K2HPO4-3H20, ph 7.2); VII. Horse serum medium, 5 hr at 4 C (control); VIII. Horse serum medium, 5 hr at room temperature; IX. Sucrose 0.3 M, Mg M, phosphate buffer 0.3 M; X. Sucrose 0.3 M, Mg M; XI. Sucrose 0.3 M, phosphate buffer 0.3 M; XII. Sucrose 0.3 M; and XIII. Mg M, phosphate buffer 0.3 M. became erratic below 0.25 M after 20 to 22 hr. However, after 48 or 72 hr incubation, maximal growth was always obtained with sucrose concentrations from 0.1 to 0.25 M. Concentrations of from 0.25 to 0.88 M were equally effective in permitting maximal growth consistently with the shortest incubation time. It was always possible to recover viable L forms up to 24 hr following suspension in 0.3 M sucrose. Glycerol concentrations of from 0.3 to 1.4 M failed to support either viability or structural integrity of the L form. Each suspension was examined by dark phase microscopy prior to inoculating its sedimented L form content into the liquid medium. Those suspensions which permitted growth after 20 to 22 hr incubation (VII-XII) displayed the intact L structure as well as many free dense large bodies, with few vacuolated or ghost forms, similar to figure 2. Those suspensions which either did not support viability or permitted growth only after prolonged incubation always contained relatively fewer dense large bodies but more vacuolated large forms. Granular material was evident within some of these otherwise empty ghost forms adhering mainly to their walllike membrane. Various degrees of L structure fragmentation as well as considerable cellular debris was always apparent. Examination of each growing culture, after 20 to 22 hr or 48 and 72 hr incubation, always revealed the typical L form morphology. Since viability of the L form could be maintained for as long as 24 hr in 0.3 M sucrose suspensions, it became of interest to determine if viability could be preserved by lyophilization in such a hypertonic solution. A large broth culture of the L form (0.8 L) was harvested at 6000 rpm after 36 hr incubation, washed twice, and resuspended in a third sample of 0.3 M sucrose. Following shell freezing, the cellular mass was lyophilized and stored at room temperature in a desiccator over P205 and NaOH. After 2 months it was still possible to obtain typical L form growth within 48 hr following inoculation of the dry powder into the liquid medium. DISCUSSION It has been indicated that initial growth of the L form in broth is difficult. That the difficulty is not due to lack of nutrients is substantiated by the fact that colonies of the L form continue to grow from pieces of agar submerged in broth. It has been suggested that the physical properties of the medium are at fault (Dienes, 1953). Our results seem partially to justify such an assumption. A definite tendency towards dispersion of the L structure was observed upon continued subculture in a liquid environment resulting in a more rapidly growing culture. Dispersion of the L structure into its intact component parts after mild sonic treatment suggests that only weak forces may play a role in maintaining the integrity of the L structure (Panos and Barkulis, 1958). A possible hypothesis for explaining the constant necessity for large inocula in broth may be the need of overcoming such external or internal forces before maximal growth can be achieved. The ability of the L form to survive suspended in various solutions is probably primarily an osmotic effect, but not all of the observations made can be readily understood in these terms. For example, suspension of the L form in equimolar concentrations of glycerol, phosphate, and sucrose was attended by a considerable difference in the viability of the organism as measured by subsequent inoculation into broth. Sucrose over a wide range of concentrations, 0.25 to 0.88 M, consistently preserved the organism in an intact and viable form. On the other hand, suspension in phosphate solutions followed by inoculation into broth gave inconsistent growth after 20 to 22 hr incubation. Suspension in glycerol was no better than suspension in water. Similar results with glycerol and sucrose have been obtained in

5 1959] VIABILITY OF STREPTOCOCCAL L FORMS 251 the preservation of mitochondria (personal communication, L. G. Abood) from mammalian tissues. However, hypertonic phosphate is toxic to mitochondria. The results with glycerol might be due to a ready penetrability of this substance into large bodies and L granules. This would prevent the maintenance of an osmotic barrier against the suspending medium. The protection of the L form over a wide range of sucrose concentrations suggests that these forms, as has been found true for mitochondria and protoplasts, can adjust rapidly to various osmotic pressures without apparent effect on their viability and function. In hypotonic solutions of sucrose or phosphate, the microscopic picture observed was of loss of structure and large body content as well as considerable cellular debris. This represents an intermediate picture between complete maintenance of form in sucrose solutions above 0.25 M and the complete destruction of the L form as observed in water suspension. Although Mg++ has been reported to stabilize protoplasts of Escherichia coli obtained by exposure to penicillin sucrose broth (Lederberg and St. Clair, 1958), Mg++ was without effect in maintaining this form. Suspensions in hypotonic solutions may result in damage or loss to some life sustaining process which is slowly repaired or replaced by the cell if removed to a nutritive and osmotically protective medium in time. This could account for the longer incubation period constantly required before maximal growth was reached with L forms from these suspensions. The structural disintegration and impairment of viability seen after suspending L forms in hypotonic and water suspensions introduces a technical difficulty in the preparation of L form material for chemical analyses. Washing with saline or water would result in only a residue portion of these forms remaining rather than an intact and viable form. Analyses of such residues would be erroneous and open to serious criticism if reported in terms of the chemical constituents of intact L forms. In a succeeding paper data will be presented documenting this point in detail. The ability to obtain viable L forms even after 24 hr suspension in hypertonic sucrose solution affords an obvious means of freeing the L form from components of the growth medium by repeated washing in sucrose. This method is being employed currently in our laboratory in various metabolic and chemical studies of L forms. The need for preserving L forms possessing definite cultural characteristics is obvious and would eliminate much of the confusion existing in reports on L forms. The ability to obtain viable L forms from lyophilized preparations even after 2 months is, therefore, worthy of note. Such preparations would allow for indefinite preservation and permit exchange of cultures with defined characteristics. The extent to which viability can be maintained in such lyophilized preparations is currently being investigated. Fragility has been reported to be a characteristic of L forms as well as a criterion for their identification. Sonic disruption investigations currently being conducted in this laboratory have demonstrated that the L form remains viable even after 23 min of exposure to 9 kc. The results of such treatment on this streptococcal L form will be presented in a subsequent publication. ACKNOWLEDGMENT We are grateful to Drs. L. Dienes and J. T. Sharp for providing cultures and making available the facilities of their laboratory to one of us (C. P.), and also to Dr. J. R. Ward and Miss S. Madoff of the same laboratory for their kind assistance. We are also indebted to Dr. J. 0. Alberts, Head, Department of Veterinary Science, University of Illinois, for our constant supply of horse serum, to Mr. N. Bartly for his assistance in obtaining photographs of the L cultures, and to our colleague Dr. J. A. Hayashi, for his helpful suggestions during the preparation of this manuscript. SUMMARY Upon prolonged subculturing in a liquid medium the macroscopic growth characteristics of the L form of the AED strain of f-hemolytic streptococcus resembled those of the parent streptococcus. The effect of solutions of varying composition and osmolarity on structural integrity and viability has been investigated. Although phosphate and glycerol solutions were either erratic or without effect, 0.25 to 0.88 M sucrose solutions were found to be osmotically protective. These results afford a means of obtaining the viable L form intact and free from components of the growth medium and suitable for chemical and metabolic studies. Successful preservation of viability of this L form for a period of 2 months by lyophilization in 0.3 M sucrose has been achieved.

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